Recombinant therapeutic proteins are widely used to treat numerous human diseases from cancer to infertility. They include various blood-clotting factors, insulin, growth hormones, enzymes, Fc fusion proteins, monoclonal antibodies and other proteins (Scott C., Bioprocess Int. 10(6): S72-S78, 2012). Many recombinant therapeutic proteins are manufactured using mammalian host cells because of the need for correct folding and post-translational modification including glycosylation. Among them, therapeutic antibodies represent one of the largest sectors of protein therapeutics with a global market of approximately $50 billion in 2011 for approximately 30 approved antibody therapeutics.
The predominant therapeutic antibodies come from antibody discovery programs that belong to four categories: chimeric antibodies, humanized antibodies, fully human antibodies from synthetic human antibody libraries selected with various display systems, and fully human antibodies from transgenic animals bearing human immunoglobulin genes (Chames P. et al, Br J Pharmacol. 157(2): 220-233, 2009). Chimeric antibodies containing human constant regions and non-human variable regions pose an immunogenicity risk in the human body and as a result have lost favor to humanized or fully human antibodies in terms of therapeutic applications. Humanized antibodies contain 90-95% human residues and 5-10% non-human residues that are essential for antigen interaction, whereas fully human antibodies contain 100% human residues. Both humanized and fully human antibodies have enjoyed great success in therapeutic applications to treat various diseases.
Development of a therapeutic antibody often takes 10-15 years including antibody discovery, engineering, production cell line development, manufacturing process development, and clinical studies. Among these tasks, antibody discovery may take 6-18 months and production cell line development may require an additional 6-10 months. One of the biggest problems with current antibody discovery methodologies is that they do not utilize the format of the final antibody product which is commonly a full-length human IgG. The selected antibodies are typically murine antibodies or fragments of human antibodies such as scFv or Fab, that require reformatting into the final IgG format before production cell line development. Reformatting sometimes leads to unexpected problems in downstream process development, including loss of activity, low expression level, aggregation, insolubility, and/or instability. Therefore further antibody engineering and optimization may be required, resulting in loss of valuable time and increased cost.
Cell line development is a critical part of the process to obtain production cell lines for any therapeutic protein including antibodies. Production cell lines should be highly productive, stable, and have correct product quality attributes including biological activity, protein sequence homogeneity, glycosylation profile, charge variants, oxidation, deamination and low levels of aggregation. CHO cells are the most popular mammalian cells for production of therapeutic proteins. Other mammalian cells like NS0 or SP2/0 cells have also been used to produce biological therapeutics (Jayapal K R, et al., Chemical Engineering Progress, 103: 40-47, 2007; Li F, et al., MAbs. 2(5): 466-477, 2010). Conventional cell line development utilizes gene amplification systems by incorporating Dihydrofolate Reductase (DHFR) or Glutamine Synthetase (GS) as selection markers. Typically up to 1000 clones are screened in a cell line development program by limiting dilution cloning in 96-well tissue culture plates. Obtaining a highly productive cell line requires gene amplification by adding selection pressure after stable transfection using, for example, Methotrexate (MTX) in the DHFR system, or Methionine Sulphoximine (MSX) in the GS system. In most cases, productivity is often the only selection criteria until a very late stage in the process when only a handful of clones are assessed for the other quality attributes important for large scale manufacturing, resulting in increased project risk and complex issues regarding downstream development.
The process of selecting a cell population of interest for use as a recombinant protein production cell line may involve expression of a cell surface or intracellular reporter molecule. High level of expression of intracellular reporters such as GFP have been shown to be cytotoxic (Liu H S, et al., Biochem Biophys Res Commun, 260: 712-717, 1999; Wallace L M, et al., Molecular Therapy Nucleic Acids. 2, e86, 2013), however, the cytotoxicity of a reporter is minimized by cell surface display.
Display techniques have been developed for high-throughput screening of proteins, such as antibodies. Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically rely on phage display, E. coli display or yeast display. Each display system has its strengths and weaknesses, however, in general these systems lack post-translational modification functions or exhibit different post-translational modification functions from mammalian cells and tend to display small antibody fragments instead of full-length IgGs. Thus, characterization of the biological activities and further development of the isolated antibody fragments often requires conversion to whole immunoglobulins and expression in mammalian cells for proper folding and post-translational processing. This conversion process may produce antibodies with binding characteristics unlike those selected for in the initial screen.
There is a need for improved processes for selection of recombinant protein producing cell lines (such as antibody-producing cell lines), wherein the selection process facilitates rapid cell line selection based on quality attributes other than productivity. The present invention provides improved compositions and methods for screening and selection of cell lines for recombinant protein production.